Replication DNA polymerases, genome instability and cancer therapies

Abstract It has been over a decade since the initial identification of exonuclease domain mutations in the genes encoding the catalytic subunits of replication DNA polymerases ϵ and δ (POLE and POLD1) in tumors from highly mutated endometrial and colorectal cancers. Interest in studying POLE and POLD1 has increased significantly since then. Prior to those landmark cancer genome sequencing studies, it was well documented that mutations in replication DNA polymerases that reduced their DNA synthesis accuracy, their exonuclease activity or their interactions with other factors could lead to increased mutagenesis, DNA damage and even tumorigenesis in mice. There are several recent, well-written reviews of replication DNA polymerases. The aim of this review is to gather and review in some detail recent studies of DNA polymerases ϵ and δ as they pertain to genome instability, cancer and potential therapeutic treatments. The focus here is primarily on recent informative studies on the significance of mutations in genes encoding their catalytic subunits (POLE and POLD1), mutational signatures, mutations in associated genes, model organisms, and the utility of chemotherapy and immune checkpoint inhibition in polymerase mutant tumors.

ity, though to varying degr ees ( 32 , 33 ). Mor e pr ecise pr estead y-sta te kinetic measurements showed that the effects on e xonuclease acti vity ranged ov er an astounding four orders of magnitude, from ∼2-fold to > 44 000-fold ( 15 , 34 ). A major outstanding question is how these mutants with drama tically dif ferent ef fects on ca talytic function can all lead to hypermutant tumors with similar mutation signatur es. Two r esidues, P286 and V411, ar e r ecurr entl y m utated, accounting for 50-75% of all POLE mutations ( 35 ). Structural studies with yeast P301R mutant enzyme, which is orthologous to human P286R, have shown that the arginine creates a positi v ely charged bulge in the exonuclease primer binding groove that prevents the correct orientation for exonucleolytic catalysis ( 36 , 37 ) (Figure 1 ). The recurrent S459F mutation also dri v es the unique muta tion signa ture and strongly reduces e xonuclease acti vity ( 31 , 34 ); howe v er, it is not located in a part of the exonuclease domain that would have an obvious effect on activity. The V411L mutation is also well away from the acti v e site and has an unknown effect on structure. The ribbon diagrams ( Figure 1 ) were made using the PDB file from ( 38 ) for the yeast enzyme and using AlphaFold ( 39 , 40 ) for the human enzyme. Domains are labeled based on ( 41 ).
Increased mutagenesis due to exonuclease domain mutations is not the only way that dysregulated POLE can contribute to increased genome instability. Decreasing the protein le v els of replication DN A pol ymerases has previousl y been shown to increase mutagenesis in yeast primarily in the form of increased DN A pol ymerase (Pol )-dependent SNVs, increased loss of heterozygosity e v ents and increased single-stranded DN A (ssDN A) (42)(43)(44)(45)(46). The increased ss-DN A likel y results from failure to repair DNA doublestrand breaks, since APOBEC3-dependent mutagenesis also increases with increasing ssDNA le v els. In cells with mutant Pol ␦, this APOBEC3-dependent mutagenesis is elevated on the lagging strand, while it is increased on both leading and lagging strands in Pol ε mutants ( 46 ). Another inter esting differ ence between Pols ␦ and ε is that low Pol ␦ le v els lead to increased breakpoints at GC-rich sequences, while reduced Pol ε does not ( 45 ). The authors provide an interesting possible explanation that Pol ␦ can potentially compensate by replicating these sequences on the leading strand under reduced Pol ε le v els, but not vice versa. Human cells have also shed light on the effects of reduced Pol ε . The Moiseeva laboratory used an auxin-inducible degron mutant allele of POLE to show that the CDC45-MCM2-7-GINS complex (CMG) can load in the absence of the Pol ε catalytic sub unit, b ut replication forks then quickl y colla pse ( 47 ). CMGs are the functional helicases used to separate double-stranded DNA during replication. The Moiseeva laboratory also found that expression of the C-terminal half of Pol ε in trans is sufficient to stabilize replication fork firing and suppress ATR activation, though fork progr ession r emains very slow. Decr easing normal Pol ε protein le v els can also dri v e replisome dysfunction and human patholo gies, including imm une deficiency and possibly tumorigenesis. Depletion of POLE in humans via homozygous or compound heterozygous mutations that cause splicing defects leads to FILS syndrome, which is characterized by facial dysmorphia, immune deficiency, li v edo and dwarfism ( 48 , 49 ). A separate set of patients with IM-AGe syndrome also have mutations affecting POLE splicing and subsequent protein le v els ( 50 ). This syndrome is char acterized by intr auterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genitourinary anomalies in males. These patients hav e variab le immune deficiencies and se v eral e v en de v eloped lymphomas, including a pediatric T-cell lymphoma. Coupled with observa tions tha t Pole4 −/ − mice, which lack a small subunit of Pol ε ( Pole4 ) that helps facilitate holoenzyme processivity, de v elop increased lymphomas ( 51 ), these observations suggest a possible link between POLE depletion and tumor de v elopment.

POLD1 MUT A TIONS IN CANCER
Altering amino acids in the exonuclease active site of Pol ␦ has long been known to induce a mutator phenotype in yeast ( 7 , 52-55 ) and to increase cancer mortality in mice ( 4 , 56 ). Mutations in the polymerase domain that decr ease r eplica tion fidelity and ribonucleotide discrimina tion ( 8 , 57 , 58 ) have also been shown to increase cancer mortality in mice ( 5 ). In humans, there are far fewer cancer driver mutations in POLD1 than in POLE . The reasons for this relati v e lack remain unclear, but some clues have emerged. Unlike in POLE , where the overwhelming majority of cancer mutations are located in the exonuclease domain, POLD1 mutations occur in the exonuclease and polymerase domains ( 15 , 16 ), which is the location of the most r ecurr ent POLD1 mutation, R689W ( 15 , 59,60 ). R689W also occurs in the DLD-1 cell line, which is a widely used epitheliallike colorectal cancer (CRC) cell line. This mutation confers a strong mutator phenotype when engineered into a yeast cell line while retaining full exonuclease activity ( 60 ). It was subsequently shown to trigger an expansion of dNTP pools tha t exacerba tes the muta tor ef fect ( 61 ). Muta tions in the POLD1 exonuclease domain do exist in human tumors and also have a high TMB ( 15,16 , 20 ). The first reported POLD1 exonuclease domain mutation, S478N, was seen inherited in a family with predisposition to CRC ( 20 ). The same study identified the P327L m utation, w hich is functionall y analogous to POLE P286L, in a different patient with CRC tumors, highlighting the conserved nature of this critical proline residue.
One possible partial explanation for the relati v e lack of POLD1 mutations in cancer may be the role for Pol ␦ in what has been called 'proofreading in trans ' ( 62 ) or 'extrinsic proofreading' ( 63 ). Pol ␦ can proofread DNA synthesis errors made by any of the three replication DNA Pols ( ␣, ␦ or ε ), w hile Pol ε can onl y proofread its own replication errors. In addition to this ability to proofread replication errors, Pol ␦ e xonuclease acti vity also plays critical roles in mismatch repair (MMR) and Okazaki fragment ma tura tion ( 64 ). Muta tions tha t compromise Pol ␦ e xonuclease acti vity might thus pr ove catastr ophic during tumor de v elopment by allowing the accumulation of mutations from DNA synthesis errors made by all three replication DN A pol ymerases instead of only failing to correct its own errors. It may then be that POLE and POLD1 mutations arise stochastically at similar rates, but the subsequent reduced fitness and decreased viability of POLD1 mutants gi v e the later appearance of a smaller number of POLD1 tumors. Of course, this Figur e 1. Ma pping cancer-associated amino acid residues to POLE structures. The yeast catalytic subunit structure ( A ) is from ( 38 ) and the predicted human catalytic subunit structure ( B ) was generated using AlphaFold ( 39 , 40 ). For each enzyme, the exonuclease domain [residues 283-488 in yeast and residues 268-473 in human ( 41 )] is shown in green and the polymerase domain (residues 554-994 in yeast and residues 539-979 in human) is shown in cyan. Ma gnified ima ges sho w only the ex onuclease domain with cancer-associa ted POLE muta tions (sho wn as red, yello w, or orange) and catalytic residues (gray) depicted as spheres in the indicated colors. D275 in humans (D290 in yeast) is both catalytic and cancer-associated in humans (*). While not a cancerassocia ted muta tion, S415 in humans (S430 in yeast) is a critical determinant of exonuclease / polymerase activity ratio and is discussed in the main text (*). The yeast structure was solved using D290A / E292A. Figures were generated using PyMOL.
has not been formally tested and other explanations are possible.

MUT A TION SIGNA TURES
POLE mutant tumors with functional MMR have a unique muta tion signa ture tha t was initially ca tegorized as a single muta tion signa tur e defined by thr ee distinct trinucleotide conte xt mutations: TCT > TAT transv ersions, TCG > TTG transitions and TTT > TGT transversions ( 65 ). These three mutations have since been computationally resolved into three distinct COSMIC mutation single base signatures (SBSs): 10a, 10b and 28, respecti v ely ( 28 ) (Figure 2 ). POLE mutant tumors lacking MMR gi v e rise to a separate muta tion signa ture, SBS14, characterized by multiple C > A transv ersions, primarily in NCT conte xts ( 28 , 30 ). A unique insertion / deletion (indel) signature was later discovered in POLE mutant tumors, consisting of an insertion of a single +A in a run of 5-10 consecuti v e As ( 66 ). Around this same time, COSMIC defined two indel signatures, ID1 and ID2 ( 28 ). ID1 is defined by insertion of a single T in homonucleotide runs of T ≥ 6 and ID2 and is essentially the same as what was observed in POLE mutant tumors ( 66 ). ID2 is defined by loss of a single T in homonucleotide runs of T ≥ 6. Samples that contained large numbers of ID1 or ID2 indels also had SBSs associated with loss of MMR alone (e.g. SBS6, SBS15) or in addition to POLE or POLD1 mutations (e.g. SBS14, SBS20) ( 28 ). Critically, the mechanisms that dri v e these different mutation spectra are poorly understood. The relati v e abundance of SBS10b TCG > TTG transitions has been shown to be highly variable in cells and tumors ( 31 ) and some have speculated that they are in fact due to replication past deaminated 5methylcytosines in CpG sequences ( 29 , 67 ).
POLD1 muta tion signa tur es have been mor e difficult to identify, in large part due to the much smaller number of POLD1 tumors and, ther efor e, fewer Pol ␦-dependent mutations. Sequencing normal cells from POLE and POLD1 carriers showed that there is increased Pol-dependent mutation burden in intestinal crypt stem cells, sperm and embryonic tissue. While these carriers do have increased risk of de v eloping cancer, they do not demonstr ate ear ly aging phenotypes ( 68 ). This same study identified two POLD1associa ted muta tion signa tures, SBS10c and SBS10d. SBS10c is characterized by enriched C > A transversions at TCT and more moderate peaks for CCT / TCA, while SBS10d is dominated by C > A at T CA / T CT. The similarities of the TCT > TAT transversions between POLD1 SBS10c / d to those in POLE SBS10a may hint at a common mechanism shared by POLE and POLD1 tumors, but the differences in the moderate peaks in POLD1 SBS10c / d and the T > G transversions in SBS28 point to likely separate and distinct mechanisms. W ha t underlies both the similarities and differences remains to be identified.

ASSOCIA TED MUT A TIONS
Mutations in PTEN and TP53 have been observed to be enriched in POLE mutated tumors. Since nearly e v ery open reading frame in these tumors has at least one mutation, a challenge has been to determine the functional significance, if any, of these associated mutations. PTEN has a welldefined function as a negati v e regulator of the PI3K-AKT pathway via its lipid phosphatase catalytic activity (69)(70)(71). Loss of PTEN function is canonically thought to dri v e tumor progression by generating sustained pro-growth signals through downstream targets. Howe v er, PTEN also has noncanonical tumor suppression roles that operate through a variety of pathways, including regulating and maintaining genome stability, heter ochr oma tin sta tes, replisome firing and coordination, and checkpoint control ( 72 , 73 ). PTEN 's role in genome stability is mediated through nuclear translocation, direct interaction with replication and repair factors, and protein phosphatase activities ( 74 , 75 ). PTEN directly colocalizes with replication foci e v en when cells are challenged with hydroxyurea (HU) ( 76 ). Disrupting PTEN interactions with replisome and fork rescue components (e.g. PCNA, MCM2, RPA1 and RAD51) triggers stalled forks and increases replication stress, suggesting that PTEN plays a critical role in fork stabilization and restart after r eplication str ess ( 76 , 77 ). Dir ectly r elated to POLE , PTENdeficient cells show increased spontaneous fork stalling and decr eased fork r estart w hen challenged with HU or a phidicolin ( 78 ). Critically, PTEN -deficient cells show reduced fork-associated RAD51, PCNA and CHEK1 after HU tr eatment. Addr essing how POLE mutant cells can deal with loss of these genome stabilizing roles and what other mutations are associated with POLE tumors will help better understand the functions of the cancer mutations.

MODEL SYSTEM DA T A
Since Pols ε and ␦ are conserved in all eukaryotes, the single-cell yeasts Sacchar om y ces cer evisiae and Schizosacchar om y ces pombe have long been excellent model systems for studying their functions ( 6-9 , 11 , 53 , 60 , 64 , 79-88 ). More recently cancer mutations have been modeled in yeast to stud y their ef fects on mutagenesis ( 24-26 , 36 , 60,61 , 67 , 89 ). One study that performed a comprehensi v e comparison of m ultiple m utant alleles showed o ver tw o orders of magnitude difference in mutation rate depending on the particular mutation examined ( 25 ). The P286R and S459F alleles (r esidues ar e noted by their orthologous human amino acid except where specifically noted) caused 150-and 30-fold incr eases in mutagenesis, r especti v ely, while the V411L allele was no different than wild type in haploid yeast. This large difference is intriguing because P286R and V411L are the two most frequent POLE mutations in cancer and tumors from both have high TMB. The same study also showed that very modest mutator alleles can cause synergistic increases when combined with defects in MMR.
Soriano et al. recently reported using S. pombe to model POLE mutations ( 67 ). As in S. cerevisiae , the P286R mutation was a strong mutator. The SBS10a hallmark T CT > TAT transversions pr edominated, with a minor fraction of SBS28-like NTT > NGT transversions. An interesting, though subtle difference was the lack of a strong TTT > TGT seen in human and mouse tumors. The largest difference, howe v er, was the complete absence of SBS10b TCG > TTG transitions, which the authors ascribe to the lack of cytosine methylation in S. pombe . They also found that S. pombe cells with mutated P286R are sensiti v e to a variety of DNA dama ging a gents, including ultraviolet (UV) light, methyl methanesulfonate and b leomy cin, suggesting a possible defect in replication fork progression rather than the sensitivity being due to a particular DNA lesion or adduct. The P286R cells are also modestly resistant to HU, which decreases dNTP concentrations, but synthetic lethal with muta tions tha t increase ov erall dNTP le v els. The presence of the P286R mutation did not raise dNTP le v els by itself, both in S. pombe ( 67 ) and in S. cerevisiae ( 37 ). They also found that knocking out Pol , a specialized translesion (TLS) DN A pol ymerase, reduced P286R-dependent mutagenesis by 6-7-fold, implicating roles for TLS-dependent synthesis and polymerase switching in POLE mutagenesis. Loss of Pol , howe v er, had no effect on mutagenesis in either yeast ( 37 , 67 ).
Recent studies using yeast have also provided insight into the possible functional consequences of V411L ( 24 , 90 ), the second most common POLE mutation in cancer. This residue is in a hairpin that is structurally at a distance from the exonuclease and polymerase active sites. Previous observa tions from dif ferent or ganisms have sho wn this mu-tation to have unique properties when compared to mutations residing within the defined exonuclease domain, like P286R ( 36 ). The V411L m utant allele has previousl y been shown to be a very weak to nonmutator in haploid yeast ( 25 ). Human tumors with V411L have slightly lower, yet still high overall, TMB than do tumors with P286R, and have weaker presence of POLE mutation signatures ( 31 ). The human enzyme with V411L was previously shown to have only modest effects on e xonuclease acti vity ( 33 ). Pellican ò et al. showed that phosphorylation of the DNA Pol ε catalytic subunit in yeast is critical for maintaining the balance between DNA synthesis and strand resection induced by replication fork stalling ( 90 ). The phosphorylated residue, S430 in S. cerevisiae , is adjacent to the ␤-hairpin loop that contains V411 (V426 in S. cerevisiae ) that mediates polymerase / exon uclease s witching in B-famil y DN A polymerases ( 91 , 92 ). Upon fork stalling, the nascent strand partitions to the exonuclease active site, driving resection. If left unchecked, this resection could hasten e v entual for k collapse. They showed that when the S430 residue is phosphorylated, partitioning to the exo site is blocked, thus reducing strand resection and helping maintain fork stability. The S430A m utant, w hich cannot be phosphorylated, also cannot block this partitioning. As a result, Pol2-S430A cells accumulate large amounts of stress-induced ssDNA and are highly sensiti v e to HU, which can be rescued b y inactiv ating e xonuclease acti vity. Barbari et al. showed that the V411L mutant is a strong m utator w hen MMR is inactivated ( 24 ). They also showed that the Pol ε enzyme with V411L mutation is an e v en more hyperacti v e polymerase on a hairpin-containing template in vitro than the P286R mutant ( 24 ). This activity is further increased when the hairpin template has a primer terminal mispair. Different exonuclease domain mutations that disrupt partitioning from the polymerase to the exonuclease site also increase mispair extension in vitro and are strong mutators in vivo ( 93 ). A new 68 amino acid motif adjacent to the catalytic core was found in yeast with homology to the same region in human POLE ( 94 ). When mutations present in this motif in human cancers were engineered into yeast Pol ε , gr oss chr omosomal rearrangements increased while having no effect on single nucleotide variant mutagenesis. These mutants also have reduced replication fork progression that was later found to be suppressed by ab lating e xonuclease acti vity ( 95 ). Taken together, recent work has pointed to both intrinsic and extrinsic factors that contribute to dri v e the unique mutagenesis found in POLE cancer mutants. One recurring theme is the importance of the balance between DNA synthesis and exonuclease activity. Tipping this balance in either direction not only has consequences for immedia te replica tion fidelity, but also appears to dri v e d ysregula tion a t the replica tion fork. To what extent this underlies the ability of POLE or POLD1 mutants to dri v e tumor de v elopment remains to be explored.

IMMUNE THERAPY APPROACHES
Immune checkpoint inhibitor (ICI) therapies have proven successful in treating solid tumors with high TMBs, especiall y w hen the high TMB is a result of MMR defi-ciencies or UV-induced DNA damage ( 96 , 97 ). Polymerase proofreading-deficient tumors have been a potential ICI target based largely on their high TMB and high degree of immune infiltr ates, particular ly CD8 + cytotoxic T-cell lymphocytes, as compared to microsatellite stable (MSS) tumors with wild-type POLE ( 98-100 ). One early report of ICI therapy efficacy in polymerase-mutated cancer was from two siblings with multifocal glioblastoma multiforme ( 101 ). The siblings both had biallelic MMR disorder and so completely lacked MMR, with whole exome sequencing showing that the glioblastoma tumors had acquired spontaneous pathogenic mutations in POLE . Both siblings had durable and prolonged responses to single-treatment nivolumab ( ␣-PD-1). In another early case report, a 57year-old woman with r ecurr ent, m ulti-thera p y-r esistant, stage III endometrial cancer with a POLE P286R mutation showed sustained response to ␣-PD-1, including regression of abdominal, retroperitoneal and pelvic deposits 7 months after follow-up ( 102 ). Prostate tumors with POLE mutations and MSS have also been reported as having a durable response to ␣-PD-1 treatment after failure of chemotherapy and surgery ( 103 ).
A mor e r ecent study used a prospecti v e cohort of POLE mutated solid tumors to test efficacy of single-treatment ␣-PD-1 ( 104 ). They restricted enrollment to specific criteria that have been used to distinguish dri v er from passenger POLE mutations (nonsynonymous POLE exonuclease mutation, high TMB, evidence of POLE mutation signature) ( 31 ), with the addition of including high immune infiltrate. They identified 12 tumors (5 CRC, 6 endometrial and 1 glioblastoma) with bona fide functional POLE mutations. Durable and significant responses were seen in se v en pa tients, and the best responding pa tients were those with high TMB, the highest fraction of POLE signa ture muta tions and higher immune infiltra tes as measured by immune deconvolution of bulk RNA sequencing data. Responses were also observ ed e xclusi v ely in MMRproficient / MSS tumors. A separa te stud y of 500 colorectal tumors included se v en POLE tumors that had generally higher le v els of tumor infiltrating lymphocytes (TILs) ( 105 ), which is consistent with the high TMB in these tumors. Howe v er, the presence of a POLE mutation with high TMB is not sufficient to attract TILs as POLE tumors had both TIL-high and TIL-low subsets. As this study did not provide the POLE variants, it would be interesting to determine whether there are any particular variants, possibly V411L, that might be overr epr esented in the TIL-low set.
The same study went further, using structural modeling to ask whether responders could be divided based on the spatial position of mutated amino acid residues. They describe three distinct spa tial loca tions, exo ca talytic site (e.g. P286), DNA binding site (e.g. S459) and neutral site (e.g. R446) (Figure 1 ), and show that m utations ma pping to the catalytic and DNA binding sites generally respond to ␣-PD-1 better than neutral site mutations. They also show that higher allelic frequencies of POLE mutations generally respond better, arguing that subclonal, and likely later occurring, mutations may have attenuated responses. One interesting observation is from the V411L tumors. While only thr ee wer e in this cohort, they defy easy categoriza-tion based on the authors' criteria. While two V411L patients showed ongoing partial responses, the patient with the largest increase from baseline in tumor size and with progressi v e disease (RECIST v1.1) while on treatment had a V411L mutation. Since the TMBs in all tumors were sufficiently high to generate the significant number of neoantigens predicted to dri v e response, simple abundance of neo-antigens is unlikely to be the cause of the variability in clinical response. The proportion of POLE -related SBS does differ, howe v er, between patient groups. The POLErelated fraction of total SBS is lower for nonresponders (1-60%) and higher for responders ( > 60%). Gi v en the recent insights into how V411L mutants may have somewhat different mechanisms than bona fide exonuclease domain mutations, it is worth considering whether this may be at least partly responsible for the varying response in these tumors.
A retrospecti v e analysis of over 14 000 pa tients a t MD Anderson Cancer Center found a significant benefit for the 68 patients with pathogenic POLE variants when treated with ICI ( 106 ). Median overall survival was 29.5 months for POLE patients with pathogenic variants (defined in the study as being annotated by InterVar or ClinVar and having peer-re vie wed citations), v ersus 11.6 months for nonpathogenic or uncertain variants. Agreeing with an earlier, smaller cohort of endometrial cancer patients ( 1 ), they show that tumors with pathogenic POLE variants confer a survi val benefit regar dless of treatment. Median survi val was not reached for these patients, versus 6.4 years for those with nonpathogenic variants. There is interesting future work to be done on POLE variants of unknown significance, as 38% had a positi v e r esponse to ICI therap y. Since the number of POLD1 mutant tumors in humans remains small, there has yet to be a larger scale measurement of ICI response in these tumors. Howe v er, a study that used CRISPR to separately engineer different POLE and POLD1 mutations into two different mouse tumor cell lines found that ICI sensitivity was significantly improved in those syngeneic POLE and POLD1 tumors ( 107 ). This raises the possibility that the benefits of ICI to POLD1 tumors may be similar to what is seen for POLE tumors.
In examining how POLE tumors respond to chemotherap y tr eatments, ther e ar e possible clues as to how POLE mutations may affect cellular responses to DNA damage. Ther e ar e now multiple case r eports of POLE patients displaying resistance to different chemotherapeutic treatments, including platinum drugs, topoisomerase-1 inhibitors and 5-fluorouracil (5-FU) ( 102,103 , 108 ). In one such case of a chemotherap y-tr ea ted pa tient with progressi v e disease, the POLE F367S exonuclease domain mutation was identified, and the patient was then switched to pembrolizumab, another ␣-PD-1 ICI. The patient subsequently had a complete response that persisted past 2 years ( 108 ). Se v eral groups have shown resistance to carbopla tin, cispla tin, 5-FU, do x orubicin and etoposide for POLE mutant cells in cultur e ( 109 , 110 ). Futur e w ork will lik ely unco ver what may be driving this increased resistance to DNA damaging agents: something intrinsic to POLE mutants, increased incidence of inactivating mutations in repair and / or signaling pathways in TMB-high POLE tumors or some other feature.

SUMMARY
The identification and characterization of cancer mutations in replication DN A pol ymerases have not only helped our understanding of mutagenesis and tumor de v elopment but also helped increase our understanding of basic replisome functions. There is much still to be learned, howe v er. After a decade of study using multiple systems, we still do not fully understand the basic mechanism behind the distinct mutation signatures in POLE and POLD1 tumors. And now the observa tions tha t many, but not all, of the highl y m utated POLE and POLD1 tumors respond to immune checkpoint therapies are poised to help us predict when certain therapies may be beneficial and when they may not. The next decade is likely to bring fresh insights into how the replisome is intimately linked to genome instability, cancer development and predicting therapeutic outcomes in patients.

SOME KEY OUTSTANDING QUESTIONS
• How do different POL mutations that show dramatically dif ferent ef fects on e xonuclease acti vity result in such similar mutation signatures in tumors and cells? • W ha t causes the relati v e lack of POLD1 mutations in tumors overall, but particularly in the exonuclease domain? • W ha t underlies the similarities and differences in the various polymerase-and MMR-associated mutation signatures? • How does the balance between polymerase and exonuclease activities contribute to mutagenesis and cancer? • W ha t factors contribute to driving POL tumor development and mutation signatures? • W ha t are the key determinants driving response (or lack thereof) to immune checkpoint therapies in POL tumors?

DA T A A V AILABILITY
No new data were generated or analyzed in support of this r esear ch.